1Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 72076 Tuebingen, Germany, 2Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen, 72074 Tuebingen, Germany, and

1Laboratory for NeuroRegeneration and Repair, Center for Neurology, Hertie Institute for Clinical Brain Research, University of Tuebingen, 72076 Tuebingen, Germany, 2Graduate School for Cellular and Molecular Neuroscience, University of Tuebingen, 72074 Tuebingen, Germany, and

Abstract

Correlative evidence suggests that GABAergic signaling plays an important role in the regulation of activity-dependent hippocampal neurogenesis and emotional behavior in adult mice. However, whether these are causally linked at the molecular level remains elusive. Nuclear factor of activated T cell (NFAT) proteins are activity-dependent transcription factors that respond to environmental stimuli in different cell types, including hippocampal newborn neurons. Here, we identify NFATc4 as a key activity-dependent transcriptional regulator of GABA signaling in hippocampal progenitor cells via an unbiased high-throughput genome-wide study. Next, we demonstrate that GABAA receptor (GABAAR) signaling modulates hippocampal neurogenesis through NFATc4 activity, which in turn regulates GABRA2 and GABRA4 subunit expression via binding to specific promoter responsive elements, as assessed by ChIP and luciferase assays. Furthermore, we show that selective pharmacological enhancement of GABAAR activity promotes hippocampal neurogenesis via the calcineurin/NFATc4 axis. Importantly, the NFATc4-dependent increase in hippocampal neurogenesis after GABAAR stimulation is required for the suppression of the anxiety response in mice. Together, these data provide a novel molecular insight into the regulation of the anxiety response in mice, suggesting that the GABAAR/NFATc4 axis is a druggable target for the therapy of emotional disorders.

Introduction

Functional and neuroimaging studies have correlated hippocampal activity, integrity, and size with emotional behavior in both humans and animals (Gilbertson et al., 2002; Campbell et al., 2004; Kitayama et al., 2005; Irle et al., 2010; Kheirbek et al., 2013). Specifically, it has been shown that impaired adult hippocampal neurogenesis is implicated in the pathogenesis of a number of cognitive and psychiatric disorders (Snyder et al., 2011; Kheirbek et al., 2012; Petrik et al., 2012; Sahay et al., 2011) and that the effective treatment of anxiety/depression-related behavior in rodents requires intact hippocampal neurogenesis (Santarelli et al., 2003; David et al., 2009; Surget et al., 2011). Importantly, the finding that adult neurogenesis occurs in the adult human hippocampus (Spalding et al., 2013) at rates comparable to those described in mice (Kempermann et al., 1997) suggests that it might as well have a functional role in the regulation of cognitive and emotional human behavior (Kheirbek and Hen, 2013). Therefore, the elucidation of the molecular link between adult hippocampal neurogenesis and emotional responses in mice might be critical for the development of more effective treatments for clinical anxiety disorders. GABA signaling, which is one of the most important regulator of activity-dependent hippocampal neurogenesis during adulthood (Ge et al., 2007), is known to play a central role in the pathophysiology of anxiety disorders (Lydiard, 2003; Nemeroff, 2003). However, it is not clear whether GABAergic modulation of adult neurogenesis affects anxiety levels in mice and which molecular players causally regulate these two events. It has been shown that GABAergic excitation shapes neural maturation and differentiation specifically by the enhancement of GABAA receptor (GABAAR) expression and activity in neural progenitor cells (Tozuka et al., 2005; Ge et al., 2006). GABAARs are hetero-oligomeric complexes composed of five subunits that are expressed and assembled in a cell type- and spatiotemporal-specific fashion in the brain. The composition of the GABAAR subunits determines their functional and pharmacological properties. Specifically, the functional expression of the α2 (GABRA2) and α4 (GABRA4) GABAAR subunits in neural progenitor cells controls adult neurogenesis in the mouse DG (Duveau et al., 2011). Vertebrate GABAAR subunits are organized in several gene clusters localized on different chromosomes originated by gene duplication. Interestingly, GABRA2 and GABRA4 subunits belong to the same genomic cluster, and this genomic organization might have been conserved to facilitate transcriptional coregulation (Joyce, 2007). However the transcriptional machinery involved in the coordinate regulation of GABAAR subunits in hippocampal neural progenitor cells still needs to be investigated.

Here, we demonstrate with several lines of evidence that GABAAR signaling operates through NFATc4 activity, an activity-dependent transcription factor expressed in hippocampal adult-born neurons (Quadrato et al., 2012). NFATc4 modulates hippocampal neurogenesis by direct transcriptional coregulation of GABRA2 and GABRA4 subunit expression by binding to specific responsive elements on their promoters. Furthermore, we show that selective pharmacological enhancement of GABAAR activity promotes hippocampal neuronal differentiation via calcineurin/NFATc4, which in turn modulates innate anxiety levels in adult mice. These findings provide a causal link between modulation of hippocampal neurogenesis and changes in anxiety levels by proposing NFATc4 as a key transcriptional regulator in GABAergic signaling and suggest that NFATc4/GABAergic signaling is a druggable target for the therapy of emotional disorders.

Materials and Methods

Animals.

Wild-type (WT) and nuclear factor of activated T cell-knock-out (NFATc4−/−) mice (Wilkins et al., 2002) were housed under standard conditions with food and water available ad libitum and kept on a 12 h dark/light cycle. Mice were maintained on a mixed C57BL/6 and 129S7 genetic background. Adult littermate male mice (between 2 and 5 months of age) were used in the study. All experiments were performed according to National Institutes of Health and European Union Animal Care standards and local animal care guidelines at the University of Tübingen.

Fluorescence intensity threshold analysis.

Quantification of the number of viable cells with a staining intensity for anti-GABRA2 or anti-GABRA4 antibodies above the “intermode” threshold was made using ImageJ software. Approximately 100 cells were counted for each coverslip. Cells above intensity threshold were divided by the total number of viable cells to calculate their percentage. Viability of cells was determined using DAPI staining.

Microarray analysis.

Hippocampal NSP cultures derived from WT and NFATc4−/− mice were differentiated for 1 d with BDNF added to the medium. Total RNA was isolated from the cells using PureLink RNA micro kit (Invitrogen) according to manufacturer's instructions. Whole transcriptome analysis was performed using an Affymetrix Mouse Gene 1.1 ST array on the Affymetrix GeneTitan System. For this purpose, triplicates of each sample were analyzed.

RNA quality was evaluated on Agilent 2100 Bioanalyzer with RNA integrity numbers (RINs) ranging from 8 to 10; all RINs >8 were considered optimal for downstream applications.

Double-stranded cDNA was synthesized from 100 ng of total RNA and subsequently linearly amplified and biotinylated using the GeneChip WT cDNA Synthesis and Amplification Kit (Affymetrix) according to the manufacturer's instructions. Fifteen micrograms of labeled and fragmented cDNA was hybridized to GeneChip Mouse Gene 1.1 ST arrays (Affymetrix). After hybridization, the arrays were washed and stained in a Fluidics Station 450 (Affymetrix) with the recommended washing procedure. Biotinylated cDNA bound to target molecules was detected with streptavidin-coupled phycoerythrin, biotinylated anti-streptavidin IgG antibodies and again streptavidin-coupled phycoerythrin according to the protocol. Arrays were scanned using the Affymetrix GeneTitan scanner and AGCC 3.0 software. Scanned images were subjected to visual inspection to check for hybridization artifacts and proper grid alignment and analyzed with Expression Console 1.0 (Affymetrix) to generate report files for quality control. Normalization of raw data was performed with Partek software, applying a robust multichip average algorithm. Hierachical cluster analysis was performed with average linkage for genes. Heat maps were generated with Bioconductor package gplots. Gene ontology classification and pathway analyses was performed using Ingenuity Pathway Analysis software (http://www.ingenuity.com/).

ChIP assay.

The ChIP assay was performed as described by Floriddia et al. (2011). Briefly, 1.5 × 105 neural progenitor cells were differentiated for 1 d in the following conditions: WT BDNF-treated, NFATc4−/− BDNF-treated, WT CsA-treated, and NFATc4−/− CSA-treated. Cells were fixed using formaldehyde solution at a final concentration of 1% for 30 min at 37°C. After cell lysis (0.5% SDS, 100 mm NaCl, 50 mm Tris HCl, pH 8.0, 5 mm EDTA), extracts were sonicated to shear DNA into 200–1000 bp lengths. Sheared chromatin solutions were incubated overnight at 4°C with rabbit monoclonal anti-NFATc4 antibody (Cell Signaling Technology) and normal rabbit IgG antibody (Cell Signaling Technology) with rotation. After incubation, the samples were immunoprecipitated with ChIP Grade Protein G Magnetic Beads (Cell Signaling Technology) and incubated for 2 h at 4°C with rotation. The precipitated complexes were washed repeatedly and incubated for 30 min at 65°C in parallel with input samples to elute the chromatin from the beads. Later, the beads were removed and samples were incubated 2 h at 65°C to reverse the crosslinks. DNA was isolated by phenol/chloroform extraction followed later by ethanol precipitation with sodium acetate. qPCR amplification of isolated DNA samples was performed as described previously using the following primers: GABRA4 forward: AGTTTGTCACTGTCACTTTGGT, GABRA4 reverse: TCCTTTCCCTTAGAAAGCATCAGA, GABRA2 forward: ACCATAGGTCAGGCTCTCCA, GABRA2 reverse: TCCTGACATTGTCCAGTCTCC, GABRA2.1 forward: ACCACAGGTGTCCAACATCAA, GABRA2.1 reverse: ATTTGAGAGGCTTCGTCAGCA. For ChIP primer design, potential NFAT-binding sites within the predicted gabra2 and gabra4 promoter regions were identified using MatInspector algorithm in Genomatix software (http://www.genomatix.de/). Each immunoprecipitation sample was normalized to input and IgG immunoprecipitation fractions for fold enrichment calculations using Ct values.

Dual luciferase reporter assay.

Hippocampal progenitor cells derived from WT and NFATc4−/− animals were seeded at a density of 1 × 104 cells in 96-well plates under the conditions of WT BDNF-treated, WT vehicle-treated, NFATc4−/− BDNF-treated, NFATc4−/− vehicle-treated, and WT CsA-treated. Cells were cotransfected with one of the LightSwitch GoClone reporter plasmids in which the luciferase expression is dependent on gabra4 or gabra2 promoter (SwitchGear Genomics) and with the Cypridina TK control construct using FuGENE HD transfection reagent. Reporter assays were performed at 24 h after transfection with LightSwitch Dual Assay system (SwitchGear Genomics) and plates were read using a luminescence plate reader (Mithras LB940 96-well plate reader; Berthold Technologies). All transfections were conducted in triplicate.

Viral vectors and stereotaxic injection.

For NFATc4 overexpression, full-length human NFATc4 cDNA (Addgene) was subcloned into a retrovial backbone containing a CAG promoter followed by internal ribosome entry site (IRES)-dsredExpress2. As a control, a CAG-IRES-dsredExpress2 vector was used. For stereotaxic injections, anesthetized (90 mg/kg ketamine and 10 mg/kg xylazine) mice were mounted on a stereotaxic alignment system (Steolting). Mice were injected unilaterally at two sites of the DG with 0.75 μl of retroviral suspension (1:5 dilution in PBS) as described previously (Gu et al., 2011). Coordinates from bregma of the first site were as follows: −2 mm anteroposterior, ±1.6 mm lateral, 2.5 mm ventral, and for the second site, −3 mm anterioposterior, ±2.6 mm lateral, 3.2 mm ventral. Animals were returned to the standard housing conditions until the desired time points after injection.

Drug treatment and BrdU labeling.

Mice were injected intraperitoneally once every 24 h with muscimol (M1523; Sigma-Aldrich) at 4 mg/kg body weight in 0.05 m HCl for 24 d. For the first 3 d of treatment, animals were also injected every 12 h with BrdU (B5002; Sigma-Aldrich) at 75 mg/kg body weight in saline. For suppression of neurogenesis, temozolomide (T2577; Sigma-Aldrich) was injected at 25 mg/kg body weight in DMSO on the first 3 d of a week for 4 weeks, as described previously (Garthe et al., 2009). For the control group, DMSO was injected following the same paradigm.

Cell counting and stereology.

For quantification of the percentage of RFP and CB double-positive cells in adult-born neurons, the percentage of RFP+/CB+ cells over the total number of RFP+ cells was assessed in the DG of all the groups. The total number of BrdU+ cells, the total number of BrdU+/CB+ cells, and the total number of BrdU+/GFAP+ cells were assessed in WT and NFATc4−/− mice treated with either muscimol or vehicle. The number of positive cells was determined in every tenth section in a series of 40 μm coronal sections spanning the complete DG using the optical dissector principle. Fluorescent signals were detected using confocal laser microscopy (LSM 510 Axiovert 200M; Zeiss). Positive cells were counted using a 63× oil objective and cells located in the upper most focal plane were not counted.

Elevated plus maze.

To test anxiety-related behavior, WT and NFATc4−/− mice between 2 and 5 months of age were tested using the elevated plus maze (EPM). Mice were placed for 5 minutes on an elevated maze composed of four arms, two with black high walls and two without walls. Each arm was 76.5 cm long and 74 cm elevated above the ground. Mice were placed in the testing room 1 h before test under dim light to allow them to habituate. The sessions were videotaped and the total distance and the distance traveled in closed and open arms were recorded using the Ethovision video-tracking system (Noldus).

Statistical analysis.

All numerical analyses were performed using Excel software (Microsoft). To compare the averages between two groups for in vitro, in vivo, and ex vivo experiments, unpaired two-tailed Student's t test was used. For the EPM test, latencies were analyzed using one-way ANOVA (with genotype as independent factor and time spent in open arm as repeated-measure factor) followed by Bonferroni's post hoc test for group comparison. Differences were considered significant at p < 0.05.

Results

After a search for activity-dependent molecular pathways in adult mouse hippocampal neural precursor cells, we identified GABAergic signaling as a central pathway targeted by NFATc4. This was achieved by carrying out an unbiased high throughput genome wide transcriptome analysis in WT versus NFATc4−/− differentiating neural precursor cells (NPCs) derived from the adult DG. The significance of gene expression changes was calculated using a t test without multiple testing corrections (Partek), selecting all transcripts with a minimum of 1.5-fold change in the expression level and p < 0.05 (the entire dataset is available upon request). Signal intensities were scaled and centered and the distance between two expression profiles was calculated using the Euclidean distance measure, which clearly shows changes between WT and NFATc4−/− gene expression profiles (Fig. 1A). Indeed, gene ontology classification and pathway analyses showed that up to 20% of all the dysregulated pathways are neuronal activity dependent (Fig. 1B). Interestingly, as opposed to other pathways, all of the differentially regulated genes belonging to the GABAR signaling show downregulation in the absence of NFATc4 expression (Fig. 1C), suggesting NFATc4 as a transcriptional node in the control of GABA signaling. Interestingly, among the members of the GABAergic signaling that were downregulated in NFATc4−/− cells, the subunits of the GABAAR α2 (GABRA2), α4 (GABRA4), and beta1 (GABRAB1) localize in the same genomic cluster on chromosome 5 (Fig. 2A), suggesting direct transcriptional coregulation of these subunits by NFATc4.

Next, we validated gene expression changes of the GABRA2, GABRA4, and GABRAB1 subunits by qRT-PCR. We found that only the GABRA2 and GABRA4 transcripts showed >2-fold downregulation (fold change ± SEM: GABRA2: 2.0 ± 0.043; GABRB1: −1.5 ± 0.2; GABRA4: 5.4 ± 0.17; Fig. 2B) in the absence of NFATc4, so these two subunits became the focus of further experiments.

Enhancement of GABAAR signaling promotes neuronal differentiation of cultured NPCs via NFATc4 activity. A, Immunofluorescence staining (red, dsred or NFATc4-dsred; green, MAP2; violet, nestin and blue DAPI) of WT, WT CsA-treated (100 ng/ml) and NFATC4−/− (−/−), and NFATC4−/− CsA-treated NPCs, differentiated for 24 h in presence of vehicle or muscimol (10 μm). Scale bar, 25 μm. B, Quantification of the number of WT NPCs, cultured for 20 h in differentiating conditions and belonging to four different populations expressing, respectively: nestin, MAP2, nestin + MAP2, or none of the two markers. Data are expressed as percentage of the total number of viable cells (DAPI staining). Approximately 100 cells were counted for each of three different coverslips. Data are shown as mean ± SEM; n = 3. C–F, Quantification of the number of WT-dsred, WT-dsred CsA-treated, NFATc4−/−-dsred, and NFATc4−/−-NFATc4-dsred NPCs cultured for 20 h in differentiating conditions and belonging to four different populations expressing, respectively: nestin, MAP2, nestin + MAP2, or none of the two markers after treatment with either vehicle or muscimol (10 μm). Data are expressed as percentage of the total number of viable cells (DAPI staining). Approximately 100 cells were counted for each of three different coverslips. Data are shown as mean ± SEM; n = 3; one-way ANOVA followed by Bonferroni post hoc test (see Table 1 for statistics).

Importantly, muscimol administration promotes the nuclear localization of NFATc4 in a subpopulation of nestin+ neural progenitor cells and not in DCX+ neuroblasts, confirming that NFATc4 is nuclear and therefore likely active at early stage of adult hippocampal progenitor cells development (Fig. 7).

To determine whether NFATc4 activity is required for GABAAR-dependent neuronal differentiation in the adult brain, we injected stereotaxically the control retrovirus expressing dsred into the DG of a group of adult WT and NFATc4−/− mice or the NFATc4-dsred expressing retrovirus in the DG of NFATc4−/− mice to selectively rescue NFATc4 expression in hippocampal progenitor cells. The three groups of mice were then injected daily for 24 d with either the GABAAR agonist muscimol (4 mg/kg) or the vehicle (Fig. 8A). We then assessed the effect of GABAergic excitation on hippocampal progenitor cells differentiation by counting the percentage of retrovirus-infected cells positive for the neuronal marker CB in the three groups of mice. Indeed, the treatment with the GABAAR agonist substantially increased the percentage of neuronal differentiation (CB+/dsred+ cells) in WT and in NFATc4−/− mice in which NFATc4 expression was rescued by retrovirus, but not in the absence of NFATc4 (mean ± SEM: WT dsred vehicle 50.8 ± 1.9%; WT dsred muscimol, 78.8 ± 2.7%; NFATc4−/− NFATc4-dsred vehicle, 49.16 ± 5.6%; NFATc4−/− NFATc4-dsred muscimol, 72.9 ± 3.76; Fig. 8B,C). Consistent with these data, we found that, 24 d after BrdU injections, the total number of BrdU+/CB+ cells increases in muscimol-treated mice compared with vehicle (mean ± SEM: WT vehicle 1102.338 ± 91.44; WT muscimol 1680.42 ± 128.80), whereas the total number of BrdU+ cells differentiated toward the glial phenotype (BrdU+/GFAP+) decreases after muscimol (mean ± SEM: WT vehicle 370.57 ± 106.7; WT muscimol 135.20 ± 69.55; Fig. 8E,F). Importantly, in NFATc4−/− mice, we observed no effect on the commitment of NPCs after muscimol administration. Moreover, despite the confirmation of a basal difference in NPC survival between WT and NFATc4−/− mice (as we have shown previously: Quadrato et al., 2012), we did not observe a difference in the total number of BrdU+ cells in muscimol-treated compared with vehicle-treated WT mice. This confirms that activation of the GABAAR/NFATc4 axis selectively affects the commitment and not the survival of NPCs (Fig. 8D,G).

Administration of GABAAR agonists, including muscimol, decreases the anxiety level in rodents (Zarrindast et al., 2001; Lippa et al., 2005). It has been also shown that increase in adult hippocampal neurogenesis after exposure to enriched environment or drug administration exerts anxiolytic effect in rodents (Santarelli et al., 2003; Schloesser et al., 2010). To elucidate whether chronic GABAergic excitation is able to decrease innate anxiety in mice via NFATc4 activation, we assessed anxiety-like behavior in WT and NFATc4−/− mice after chronic administration of muscimol. Importantly, we have reported previously that NFATc4−/− mice show no differences in basal anxiety level or locomotor performance and that their behavioral impairment is restricted only to the formation of spatial long-term memory (Quadrato et al., 2012). Therefore, NFATc4−/− mice are suitable for studying the modulation of anxiety level after chronic GABAAR stimulation. As a behavioral read out of anxiety levels, we used the EPM, a classical rodent model of anxiety used for testing anxiogenic and anxiolitic compounds (Hogg, 1996). WT and NFATc4−/− mice were injected daily for 24 d with either muscimol (4 mg/kg) or vehicle and then 24 h after the last injection tested in the EPM (Fig. 9A). Consistent with previous data (Quadrato et al., 2012), we found no difference in time spent in open arms between vehicle-injected WT and NFATc4−/− mice (mean ± SEM: WT vehicle, 4.12 ± 1.95%; NFATc4−/− vehicle, 18.06 ± 3.97%; Fig. 9A–C), but, as predicted, we observed a significant increase in the time that WT mice spent in open arms in response to muscimol administration (mean ± SEM: WT vehicle, 4.12 ± 1.95%; WT muscimol, 57.02 ± 10.06%; Fig. 9A–C). Similarly, we found that, consistent with a reduction in anxiety levels, muscimol-treated mice showed a tendency to spend more time in the crossing area and to travel more compared with vehicle-treated mice (Fig. 9E,F). Importantly, this effect was not observed in NFATc4−/− mice (mean ± SEM: NFATc4−/− vehicle, 18.06 ± 3.97% NFATc4−/− muscimol, 28.28 ± 3.23%; Fig. 9A–C), confirming that GABAAR stimulation decreases innate anxiety in mice via NFATc4 activation.

GABAergic signaling regulates innate anxiety by enhancing adult hippocampal neurogenesis via NFATc4. A, Diagram summarizing the experimental design. B, Track trace of the path traveled inside the EPM (as a measure of anxiety-related behavior) by adult WT and NFATc4−/− mice injected intraperitoneally with either vehicle or muscimol and adult WT mice injected with TMZ or TMZ + muscimol. Shown is the percentage of time spent in open arms (C), the percentage of time spent in the closed arms (D), and the percentage of time spent in the crossing area (E) of the EPM for each group of mice. F, Distance traveled (in centimeters) by each group of mice. Data are shown as mean ± SEM; n = 8–9; one-way ANOVA followed by Bonferroni post hoc test: *p < 0.05; **p < 0.01; ***p < 0.001. G, Immunofluorescence staining BrdU (green) and DAPI (blue) of cells in the DG of adult mice after treatment with either DMSO or TMZ for 4 weeks. BrdU (75 mg/kg) was injected twice daily for 3 d at the beginning of the TMZ injection. Scale bar, 100 μm. H, Track trace of the path traveled inside the EPM as a measure of anxiety-related behavior by adult mice injected intraperitoneally with either vehicle or muscimol and tested 4 and 24 h after the injection. I, Percentage of time spent in open arms of EPM for each group of mice Data are shown as mean ± SEM; n = 8–9; one-way ANOVA followed by Bonferroni post hoc test: *p < 0.05.

To investigate the contribution of adult hippocampal neurogenesis in the NFATc4-mediated modulation of anxiety levels, we ablated adult neurogenesis in mice after GABAAR stimulation by using the DNA-alkylating agent temozolomide (TMZ), as described previously (Garthe et al., 2009). We subjected the mice injected with muscimol or vehicle to treatment with TMZ (25 mg/kg) for 3 d every week for 4 weeks (Fig. 9A). After chronic administration of both muscimol and TMZ, we did not observe any weight loss or behavioral abnormality. To confirm the suppression of neurogenesis after TMZ treatment, BrdU (75 mg/kg) was injected for the first 3 d twice per day and BrdU staining was performed 4 weeks after injection. Consistent with previous data by Garthe et al. (2009), the number of BrdU+ cells in the DG decreased by >80% (Fig. 9) in TMZ-treated mice compared with vehicle. Importantly, we found that, after depletion of the pool of newborn neurons, the anxiolytic effect of muscimol was blocked (mean ± SEM: WT TMZ, 18.74 ± 2.67%; WT TMZ muscimol, 30.83 ± 5.6%; Fig. 9B,C), suggesting that NFATc4-dependent increase in neurogenesis is required for the anxiolytic effect observed in mice after GABAAR stimulation. To demonstrate that the anxiolytic effects observed are consistent with the time course required for neurogenesis, we tested the behavior of the mice in the EPM task 4 and 24 h after a single dose injection of muscimol. We observed an increase in the time spent in the open arms after 4 h but, as opposed to chronic administration, the anxyolitic effect of muscimol was not observed at the 24 h time points (Fig. 9H,I). These data suggest that, although the acute administration of muscimol has an effect on other brain regions, its chronic anxiolytic effect is mediated via increased neurogenesis.

Several studies suggest that gene expression levels of the different subunits of the GABAR change in response to extracellular stimuli (Tseng et al., 1994; Harris et al., 1995). However, despite the fact that GABA is the most important inhibitory transmitters at CNS synapses and that alterations in GABAAR expression levels are implicated in the pathogenesis of several CNS disorders including anxiety (Möhler, 2006), little was known about the transcriptional regulation of the GABAAR subunits.

Our data show that, in the absence of NFATc4, BDNF fails to stimulate the GABRA2 and GABRA4 promoters, leading to downregulation of GABRA2 and GABRA4 gene and protein expression in NPCs. In addition, we found that NFATc4 occupies specific transcriptional binding sites on GABRA2 and GABRA4 promoters and drives their expression, as shown by the ChIP and luciferase assays, respectively. Furthermore, NFATc4 rescue in null NPCs restores GABRA2 and GABRA4 expression levels, further confirming NFATc4 as a major player in the transcriptional machinery regulating GABRAAR expression.

NFAT-mediated transcriptional regulation is orchestrated, in many cases, by the formation of composite regulatory complexes (Macián et al., 2001; Soto-Nieves et al., 2009). Indeed, via genome-wide analysis, we have identified a number of transcription factors dysregulated in NFATc4−/− NPCs, including the four members of the early growth responsive (EGR) protein family (Table 2). EGR3 in particular has been shown to regulate GABRA4 expression through direct binding to its promoter after BDNF administration (Roberts et al., 2005). Similarly, through bioinformatics analysis, we have identified single and composite potential responsive elements for NFAT and other activity-dependent transcription factors, including EGRs, in the promoter region of the GABRA2 and GABRA4 genes (data not shown). These observations suggest that NFATc4 may control GABAergic signaling transcriptionally in hippocampal NPCs by regulating other activity-dependent transcription factors and by forming composite enhancer complexes on the GABRA2 and GABRA4 genomic cluster.

We also demonstrate here that calcineurin activity is required for NFATc4-dependent regulation of GABRAAR in hippocampal NPCs. Indeed, CsA-mediated inhibition of calcineurin activity blocks NFATc4 binding to the GABRA2 and GABRA4 promoters and leads to a decrease in their expression, phenocopying NFATc4 absence.

The major cellular event that leads to calcineurin activation is an increase in intracellular calcium (Rusnak and Mertz, 2000). It is known that GABAergic depolarization enhances Ca2+ levels, mainly via activation of Ca2+ channels and via release of Ca2+ from internal stores (Ge et al., 2007; Young et al., 2010). Along the same lines, in our study, we show that enhancement of GABAergic transmission in NPCs leads to increases in NFATc4 transcriptional activity via calcineurin activation.

GABAergic depolarization after the administration of GABAAR agonists increases Ca2+ levels in hippocampal neural progenitor cells, promoting activity-dependent neuronal differentiation (Tozuka et al., 2005; Ge et al., 2006). These findings are consistent with the present study, in which we found that administration of a selective agonist of GABAAR increases the percentage of new neurons generated from hippocampal progenitor cells both in vitro and in vivo. The same treatment did not produce any effect in NFATc4−/− mice, but, importantly, selective NFATc4 rescue of function in hippocampal NPCs derived from null mice restores neuronal differentiation to the WT level after muscimol administration. Interestingly, we have shown that the GABAAR/NFATc4 axis is selectively active in a subpopulation of early neural stem/progenitor cells, suggesting a transient effect of NFATc4. Indeed, the nuclear translocation of NFATc4 induced by GABAergic excitation occurs only in a restricted time frame that is critical for neural stem/progenitor cells fate choice. These data suggest a major role for NFATc4 in promoting neuronal differentiation in adult hippocampal progenitor cells after GABAergic excitation.

In the last several years, there has been speculation about the role of adult-born hippocampal neurons in the regulation of cognitive and emotional response. Although the hypothesis that a decrease in hippocampal neurogenesis per se might result in depression or anxiety has generally been rejected (Petrik et al., 2012), increasing evidence suggests that adult-born neurons play an important role in both discrimination of similar memory traces (pattern separation; Aimone et al., 2006; Dupret et al., 2008; Clelland et al., 2009; Deng et al., 2010; Sahay et al., 2011) and in buffering stress and depressive behavior (Snyder et al., 2011). Specifically, the current standing of the neurogenesis hypothesis on emotional and depressive disorders infers that the treatment of anxiety/depression-related behavior in mice requires intact hippocampal neurogenesis (Santarelli et al., 2003; David et al., 2009; Surget et al., 2011). Furthermore, increases in adult neurogenesis after physiological stimuli that drive changes in neuronal activity, including exposure to enriched environment and running, is associated with a rescue of anxiety-like phenotype (Schloesser et al., 2010; Onksen et al., 2012).

Similarly, we found that increased chronic GABAergic excitation decreases the anxiety response in WT mice, but not in in NFATc4−/− mice, in which the hippocampal neurogenic response is impaired. Importantly, TMZ-dependent ablation of neurogenesis phenocopied NFATc4−/− mice, further suggesting that an increase in adult hippocampal neurogenesis is required for the anxiolytic effect of GABAAR stimulation. Although our data demonstrate the essential role of hippocampal adult-born neurons in controlling anxiety, future studies are needed to elucidate the crosstalk between adult hippocampal neurogenesis and the function of other well characterized limbic structures involved in the anxiety response.

In summary, we have identified a positive excitatory feedback loop between GABAergic signaling and NFATc4 in which stimulation of GABAAR raises calcineurin/NFATc4 transcriptional activity, leading to increased adult hippocampal neurogenesis (Fig. 10A). These results provide novel functional and molecular insights into the regulation of anxiety, thus highlighting the therapeutic potential of the modulation of adult hippocampal neurogenesis via the GABAAR/NFATc4 axis in emotional disorders.

Footnotes

This work was supported by the Hertie Foundation and the Deutsche Forschungsgemeinschaft (S.D.G.) and the Fortune Program of the University of Tübingen (G.Q.). We thank Tuan Nguyen for important conceptual feedback, Alexandra Lepier for the design and the production of the retroviruses used, Maria Mina for technical help, Jeffery D. Molkentin for providing the NFATc4-null mice, and Verdon Taylor for providing the Hes5-GFP mice.

The authors declare no competing financial interests.

Correspondence should be addressed to either of the following: Giorgia Quadrato,
Molecular Neuroregeneration, Division of Brain Sciences, Department of Medicine, Imperial College London, London W12 ONN, United Kingdom,giorgia.quadrato{at}uni-tuebingen.de; or Simone Di Giovanni,
Molecular Neuroregeneration, Division of Brain Sciences, Department of Medicine, Imperial college London, London W12 ONN, United Kingdom,s.di-giovanni{at}imperial.ac.uk